MurC gene and enzyme of Pseudomonas aeruginosa

ABSTRACT

This invention provides isolated polynucleotides that encode the MurC protein of  Pseudomonas aeruginosa . Purified and isolated MurC recombinant proteins are also provided. Nucleic acid sequences which encode functionally active MurC proteins are described. Assays for the identification of modulators of the of expression of murC and inhibitors of the activity of MurC, are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/154,073, filed Sep. 14, 1999, and is a National Stage Filing ofPCT/US00/24845, having an International Filing Date of Sep. 11, 2000,the contents of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

This invention relates to the genes and enzymes involved in cell wallsynthesis in bacteria, and particularly to the inhibition of suchenzymes.

BACKGROUND OF THE INVENTION

The emegence of mluti-drug resistant bacteria has led to an increaseddemand for new antibiotics with new modes of action. The biosyntheticpathway of the bacterial cell wall contains several attractive targets.Some of the enzymes in that pathway are proven targets for antibioticssuch as β-lactams and glycopeptides antibiotics.

The bacterial cell wall is a polymer—a single molecule composed ofpeptidoglycan—that defines the boundary and shape of the cell. Assembledby crosslinking glycan chains with short peptide bridges (Rogers, H. J.,H. R. Perkins, and J. B. Ward, 1980, Biosynthesis of peptidoglycan. p.239–297. In Microbial cell walls and membranes. Chapman & Hall Ltd.London), the completed structure is strong enough to maintain cellintegrity against an osmotic pressure differential of over fouratmospheres, but also flexible enough to allow the cell to move, growand divide.

The construction of the peptidoglycan begins in the cytoplasm with anactivated sugar molecule, UDP-N-acetylglucosamine. After two reactions(catalyzed by MurA and MurB) that result in the placement of a lactylgroup on the 3-OH of the glucosamine moiety, a series of ATP-dependentamino acid ligases (MurC, -D, -E, and -F) catalyze the stepwisesynthesis of the pentapeptide sidechain using the newly synthesizedlactyl carboxylate as the first acceptor site. After attachment of thesugar pentapeptide to a lipid carrier in the plasma membrane, anotherglucosamine unit is added to the 4-OH of the muramic acid moiety. Thecompleted monomeric building block is moved across the membrane into theperiplasm where the penicillin-binding proteins enzymatically add itinto the growing cell wall (Lugtenberg, E. J. J., 1972, Studies onEscherichia coli enzymes involved in the synthesis of UridineDiphosphate-N-Acetyl-Muramyl-pentapeptide. J. Bacteriol. 110:26–34;Mengin-Lecreulx, D., B. Flouret, and J. van Heijenoort, 1982,Cytoplasmic steps of peptidoglycan synthesis in Escherichia coli. J.Bacteriol. 151: 1109–1117).

Among the potential enzyme targets involved in cell wall biosynthesis isMurC, UDP-N-acetylmuramoyl ligase. This enzyme catalyses theATP-dependent addition of L-alanine to UDP-N-acetylmuramoyl to form theprecursor UDP-N-acetylmuramoyl-L-alanine. This step is essential forcell wall formation in both Gram (−ve) and Gram (+ve) bacteria. Thus,inhibitors of this enzyme are likely broad spectrum antibiotics.

SUMMARY OF THE INVENTION

Polynucleotides and polypeptides of Pseudomonas aeruginosa MurC, anenzyme involved in bacterial cell wall biosynthesis are provided. Therecombinant MurC enzyme is catalytically active in ATP-dependentD-glutamate addition reactions. The enzyme is used in in vitro assays toscreen for antibacterial compounds that target cell wall biosynthesis.The invention includes the polynucleotides, proteins encoded by thepolynucleotides, and host cells expressing the recombinant enzyme,probes and primers, and the use of these molecules in assays.

An aspect of this invention is a polynucleotide having a sequenceencoding a Pseudomonas aeruginosa MurC protein, or a complementarysequence. In a particular embodiment the encoded protein has a sequencecorresponding to SEQ ID NO:2. In other embodiments, the encoded proteincan be a naturally occurring mutant or polymorphic form of the protein.In preferred embodiments the polynucleotide can be DNA, RNA or a mixtureof both, and can be single or double stranded. In particularembodiments, the polynucelotide is comprised of natural, non-natural ormodified nucleotides. In some embodiments, the internucleotide linkagesare linkages that occur in nature. In other embodiments, theinternucleotide linkages can be non-natural linkages or a mixture ofnatural and non-natural linkages. In a most preferred embodiment, thepolynucleotide has a sequence shown in SEQ ID NO:1.

An aspect of this invention is a polynucleotide having a sequence of atleast about 25 contiguous nucleotides that is specific for a naturallyoccurring polynucleotide encoding a Pseudomonas aeruginosa MurC protein.In particular preferred embodiments, the polynucleotides of this aspectare useful as probes for the specific detection of the presence of apolynucleotide encoding a Pseudomonas aeruginosa MurC protein. In otherparticular embodiments, the polynucleotides of this aspect are useful asprimers for use in nucleic acid amplification based assays for thespecific detection of the presence of a polynucleotide encoding aPseudomonas aeruginosa MurC protein. In preferred embodiments, thepolynucleotides of this aspect can have additional components including,but not limited to, compounds, isotopes, proteins or sequences for thedetection of the probe or primer.

An aspect of this invention is an expression vector including apolynucleotide encoding a Pseudomonas aeruginosa MurC protein, or acomplementary sequence, and regulatory regions. In a particularembodiment the encoded protein has a sequence corresponding to SEQ IDNO:2. In particular embodiments, the vector can have any of a variety ofregulatory regions known and used in the art as appropriate for thetypes of host cells the vector can be used in. In a most preferredembodiment, the vector has regulatory regions appropriate for theexpression of the encoded protein in gram-negative prokaryotic hostcells. In other embodiments, the vector has regulatory regionsappropriate for expression of the encoded protein in gram-positive hostcells, yeasts, cyanobacteria or actinomycetes. In some preferredembodiments the regulatory regions provide for inducible expressionwhile in other preferred embodiments the regulatory regions provide forconstitutive expression. Finally, according to this aspect, theexpression vector can be derived from a plasmid, phage, virus or acombination thereof.

An aspect of this invention is host cell comprising an expression vectorincluding a polynucleotide encoding a Pseudomonas aeruginosa MurCprotein, or a complementary sequence, and regulatory regions. In aparticular embodiment the encoded protein has a sequence correspondingto SEQ ID NO:2. In preferred embodiments, the host cell is a yeast,gram-positive bacterium, cyanobacterium or actinomycete. In a mostpreferred embodiment, the host cell is a gram-negative bacterium.

An aspect of this invention is a process for expressing a MurC proteinof P. aeruginosa in a host cell. In this aspect a host cell istransformed or transfected with an expression vector including apolynucleotide encoding a Pseudomonas aeruginosa MurC protein, or acomplementary sequence. According to this aspect, the host cell iscultured under conditions conducive to the expression of the encodedMurC protein. In particular embodiments the expression is inducible orconstitutive. In a particular embodiment the encoded protein has asequence corresponding to SEQ ID NO:2.

An aspect of this invention is a purified polypeptide having an aminoacid sequence of SEQ ID NO:2 or the sequence of a naturally occurringmutant or polymorphic form of the protein.

An aspect of this invention is a method of determining whether acandidate compound can inhibit the activity of a P. aeruginosa MurCpolypeptide. According to this aspect a polynucleotide encoding thepolypeptide is used to construct an expression vector appropriate for aparticular host cell. The host cell is transformed or transfected withthe expression vector and cultured under conditions conducive to theexpression of the MurC polypeptide. The cell is contacted with thecandidate. Finally, one measures the activity of the MurC polypeptide inthe presence of the candidate. If the activity is lower relative to theactivity of the protein in the absence of the candidate, then thecandidate is a inhibitor of the MurC polypeptide. In preferredembodiments, the polynucleotide encodes a protein having an amino acidsequence of SEQ ID NO:2 or a naturally occurring mutant of polymorphicform thereof. In other preferred embodiments, the polynucleotide has thesequence of SEQ ID NO:1. In particular embodiments, the relativeactivity of MurC is determined by comparing the activity of the MurC ina host cell. In some embodiments, the host cell is disrupted and thecandidate is contacted to the released cytosol. In other embodiments,the cells can be disrupted contacting with the candidate and beforedetermining the activity of the MurC protein. Finally, according to thisaspect the relative activity can determined by comparison to apreviously measured or expected activity value for the MurC activity inthe host under the conditions. However, in preferred embodiments, therelative activity is determined by measuring the activity of the Mur Cin a control cell that was not contacted with a candidate compound. Inparticular embodiments, the host cell is a pseudomonad and the proteininhibited is the MurC produced by the pseudomonad.

An aspect of this invention is a compound that is an inhibitor of a P.aeruginosa MurC protein an assay described herein. In preferredembodiments, the compound is an inhibitor of a P. aeruginosa MurCprotein produced by a host cell comprising an expression vector of thisinvention. In most preferred embodiments, the compound is also aninhibitor of MurC protein produced by a pathogenic strain P. aeruginosaand also inhibits the growth of said pseudomonad.

An aspect of this invention is a pharmaceutical preparation thatincludes an inhibitor of P. aeruginosa MurC and a pharmaceuticallyacceptable carrier.

An aspect of this invention is a method of treatment comprisingadministering a inhibitor of the P. aeruginosa MurC to a patient. Thetreatment can be prophylactic or therapeutic. In preferred embodiments,the appropriate dosage for a particular patient is determined by aphysician.

By “about” it is meant within 10% to 20% greater or lesser thanparticularly stated.

As used herein an “inhibitor” is a compound that interacts with andinhibits or prevents a polypeptide of MurC from catalyzing theATP-dependent addition of L-alanine to UDP-N-acetylmuramoyl precursor.

As used herein a “modulator” is a compound that interacts with an aspectof cellular biochemistry to effect an increase or decrease in the amountof a polypeptide of MurC present in, at the surface or in the periplasmof a cell, or in the surrounding serum or media. The change in amount ofthe MurC polypeptide can be mediated by the effect of a modulator on theexpression of the protein, e.g., the transcription, translation,post-translational processing, translocation or folding of the protein,or by affecting a component(s) of cellular biochemistry that directly orindirectly participates in the expression of the protein. Alternatively,a modulator can act by accelerating or decelerating the turnover of theprotein either by direct interaction with the protein or by interactingwith another component(s) of cellular biochemistry which directly orindirectly effects the change.

All of the references cited herein are incorporated by reference intheir entirety as background material.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A & 1B. Nucleotide sequence (SEQ ID NO: 1) and the predictedamino acid sequence (SEQ ID NO:2) of P. aeruginosa murC. The amino acidsequence (SEQ ID NO:2) is presented in three-letter code below thenucleotide sequence (nucleotides 59 to 1520 of SEQ ID NO: 1).

FIG. 2. Production of recombinant P. aeruginosa MurC.

Lane 1, Molecular weight markers; Lane 2, IPTG-induced lysate of cells(BL21(DE3)/pLysS) containing the control vector pET-15b; Lane 3,uninduced cell lysate containing the control vector pET-15b; lane 4,column-purified MurC; Lane 5 IPTG-induced lysate of cells expressingMurC; Lane 6, uninduced lysate of cells containing murC.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides polynucleotides and polypeptides of a cell wallbiosynthesis gene from Pseudomonas aeruginosa, referred to herein asMurC. The polynucleotides and polypeptides are used to further provideexpression vectors, host cells comprising the vectors, probes andprimers, antibodies against the MurC protein and polypeptides thereof,assays for the presence or expression of MurC and assays for theidentification of modulators and inhibitors of MurC.

Bacterial MurC, UDP-N-acetylmuramyl:L-alanine ligase, a cytoplasmicpeptidoglycan biosynthetic enzyme, catalyzes the ATP-dependent additionof L-alanine to the UDP-N-acetylmuramyl precursor, generating theUDP-N-acetylmuramoyl-L-alanine.

The murC gene was cloned from Pseudomonas aeruginosa. Sequence analysisof the P. aeruginosa murC gene revealed an open reading frame of 487amino acids. The deduced amino acid sequence of P. aeruginosa MurC ishomologous to MurC from Escherichia coli, Haemophilus influenza,Bacillus subtilis and S. aureus. Recombinant MurC protein from P.aeruginosa was over-produced as His-tagged fusion protein in Escherichiacoli host cells and the enzyme was purified to apparent homogeneity. Therecombinant enzyme catalyzed the ATP-dependent addition of L-alanine tothe UDP-N-acetylmuramyl precursor.

Nucleic acids encoding murC from Pseudomonas aeruginosa are useful inthe expression and production of the P. aeruginosa MurC protein. Thenucleic acids are also useful in providing probes for detecting thepresence of P. aeruginosa.

Polynucleotides

Polynucleotides useful in the present invention include those describedherein and those that one of skill in the art will be able to derivetherefrom following the teachings of this specification. A preferredaspect of the present invention is an isolated nucleic acid encoding aMurC protein of Pseudomonas aeruginosa. A preferred embodiment is anucleic acid having the sequence disclosed in FIG. 1, SEQ ID NO:1 anddisclosed as follows:

CTCCATGGCA GACCAGGCAC GCAGCCTGGC GAAACCCGAG GCTACCCGGA (SEQ ID NO:1)CGGTGGTCGA TGCCTGCCTG GAGGTGGCCC GTGGTTAAAG AACCGAATGG CGTCACCCGGACCATGCGCC GTATCCGCCG CATCCATTTC GTCGGCATCG GCGGCGCCGG TATGTGCGGGATCGCCGAAG TGCTGCTGAA CCTCGGCTAC GAGGTATCCG GCTCGGACCT CAAGGCCTCGGCGGTGACCG AGCGCCTGGA GAAGTTCGGC GCGCAGATCT TCATCGGCCA CCAGGCGGAAAACGCCGACG GCGCCGACGT GCTGGTGGTG TCCAGTGCCA TCAACCGGGC CAACCCGGAAGTGGCATCGG CCCTGGAACG GCGGATTCCG GTGGTGCCGC GTGCGGAGAT GCTCGCCGAGCTGATGCGCT ACCGGCACGG CATCGCGGTA GCCGGCACCC ACGGCAAGAC CACCACTACCAGCCTGATCG CCTCGGTGTT CGCCGCCGGC GGCCTGGACC CGACCTTCGT CATCGGCGGCCGGCTGAACG CCGCCGGGAC CAACGCCCAG CTCGGCGCCA GCCGCTACCT GGTGGCCGAGGCCGACGAGA GCGACGCCAG CTTCCTGCAC CTGCAACCGA TGGTCGCGGT GGTCACCAATATCGACGCCG ACCACATGGC GACCTACGGC GGCGACTTCA ACAAGCTGAA GAAGACCTTCGTCGAGTTCC TCCACAACCT GCCGTTCTAC GGACTGGCGG TGATGTGCGT GGATGATCCGGTTGTGCGTG AGATCCTCCC GCAGATCGCC CGCCCGACCG TGACCTACGG CCTCAGCGAAGACGCCGACG TGCGCGCGAT CAACATCCGC CAGGAAGGCA TGCGCACCTG GTTCACCGTGTTGCGCCCGG AGCGCGAGCC GCTGGACGTC TCGGTGAACA TGCCCGGCCT GCACAACGTGCTGAATTCCC TGGCGACCAT CGTCATCGCT ACCGACGAGG GCATCTCCGA CGAAGCCATCGTCCAGGGGC TGTCCGGCTT CCAGGGCGTA GGCCGGCGCT TCCAGGTCTA CGGCGAGCTGCAGGTCGAGG GTGGCAGCGT GATGCTGGTG GACGATTACG GCCACCATCC GCGCGAAGTCGCCGCGGTGA TCAAGGCGAT CCGTGGCGGT TGGCCGGAGC GTCGCCTGGT GATGGTCTACCAGCCGCATC GCTATACCCG TACCCGCGAC CTGTACGAAG ACTTCGTGCA GGTGCTGGGCGAAGCCAACG TGCTGCTGTT GATGGAGGTC TATCCGGCCG GCGAAGAGCC GATCCCGGGAGCCGACAGCC GCCAGCTGTG CCACAGCATC CGCCAGCGCG GCCAGCTTGA CCCGATCTACTTCGAGCGCG ACGCCGACCT GGCGCCGCTG GTCAAGCCGC TGCTGCGCGC TGGCGACATCCTGCTTTGCC AGGGCGCTGG CGATGTCGGC GGCCTGGCCC CGCAACTGAT CAAGAACCCGCTGTTCGCCG GCAAGGGAGG GAAGGGCGCA TGAACCTTTG CCTCGATAGC CTGCTGAACG

The translation initiation and termination codons are underlined.

The isolated nucleic acid molecule of the present invention can includea ribonucleic or deoxyribonucleic acid molecule, which can be single(coding or noncoding strand) or double stranded, as well as syntheticnucleic acid, such as a synthesized, single stranded polynucleotide.

The present invention also relates to recombinant vectors andrecombinant hosts, both prokaryotic and eukaryotic, which contain thesubstantially purified nucleic acid molecules disclosed throughout thisspecification.

As used herein a “polynucleotide” is a nucleic acid of more than onenucleotide. A polynucleotide can be made up of multiple polynucleotideunits that are referred to by description of the unit. For example, apolynucleotide can comprise within its bounds a polynucleotide(s) havinga coding sequence(s), a polynucleotide(s) that is a regulatory region(s)and/or other polynucleotide units commonly used in the art.

An “expression vector” is a polynucleotide having regulatory regionsoperably linked to a coding region such that, when in a host cell, theregulatory regions can direct the expression of the coding sequence. Theuse of expression vectors is well known in the art. Expression vectorscan be used in a variety of host cells and, therefore, the regulatoryregions are preferably chosen as appropriate for the particular hostcell.

A “regulatory region” is a polynucleotide that can promote or enhancethe initiation or termination of transcription or translation of acoding sequence. A regulatory region includes a sequence that isrecognized by the RNA polymerase, ribosome, or associated transcriptionor translation initiation or termination factors of a host cell.Regulatory regions that direct the initiation of transcription ortranslation can direct constitutive or inducible expression of a codingsequence.

Polynucleotides of this invention contain full length or partial lengthsequences of the MurC gene sequences disclosed herein. Polynucleotidesof this invention can be single or double stranded. If single stranded,the polynucleotides can be a coding, “sense,” strand or a complementary,“antisense,” strand. Antisense strands can be useful as modulators ofthe gene by interacting with RNA encoding the MurC protein. Antisensestrands are preferably less than full length strands having sequencesunique or specific for RNA encoding the protein.

The polynucleotides can include deoxyribonucleotides, ribonucleotides ormixtures of both. The polynucleotides can be produced by cells, incell-free biochemical reactions or through chemical synthesis.Non-natural or modified nucleotides, including inosine, methyl-cytosine,deaza-guanosine, etc., can be present. Natural phosphodiesterinternucleotide linkages can be appropriate. However, polynucleotidescan have non-natural linkages between the nucleotides. Non-naturallinkages are well known in the art and include, without limitation,methylphosphonates, phosphorothioates, phosphorodithionates,phosphoroamidites and phosphate ester linkages. Dephospho-linkages arealso known, as bridges between nucleotides. Examples of these includesiloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, andthioether bridges. “Plastic DNA,” having, for example, N-vinyl,methacryloxyethyl, methacrylamide or ethyleneimine internucleotidelinkages, can be used. “Peptide Nucleic Acid” (PNA) is also useful andresists degradation by nucleases. These linkages can be mixed in apolynucleotide.

As used herein, “purified” and “isolated” are utilized interchangeablyto stand for the proposition that the polynucleotide, protein andpolypeptide, or respective fragments thereof in question have beenremoved from the in vivo environment so that they exist in a form orpurity not found in nature. Purified or isolated nucleic acid moleculescan be manipulated by the skilled artisan, such as but not limited tosequencing, restriction digestion, site-directed mutagenesis, andsubcloning into expression vectors for a nucleic acid fragment as wellas obtaining the wholly or partially purified protein or proteinfragment so as to afford the opportunity to generate polyclonalantibodies, monoclonal antibodies, or perform amino acid sequencing orpeptide digestion. Therefore, the nucleic acids claimed herein can bepresent in whole cells or in cell lysates or in a partially orsubstantially purified form. It is preferred that the molecule bepresent at a concentration at least about five-fold to ten-fold higherthan that found in nature. A polynucleotide is considered substantiallypure if it is obtained purified from cellular components by standardmethods at a concentration of at least about 100-fold higher than thatfound in nature. A polynucleotide is considered essentially pure if itis obtained at a concentration of at least about 1000-fold higher thanthat found in nature. We most prefer polynucleotides that have beenpurified to homogeneity, that is, at least 10,000–100,000 fold. Achemically synthesized nucleic acid sequence is considered to besubstantially purified when purified from its chemical precursors by thestandards stated above.

Included in the present invention are assays that employ further novelpolynucleotides that hybridize to P. aeruginosa murf sequences understringent conditions. By way of example, and not limitation, a procedureusing conditions of high stringency is as follows: Prehybridization offilters containing DNA is carried out for 2 hr. to overnight at 65° C.in buffer composed of 6×SSC, 5× Denhardt's solution, and 100 μg/mldenatured salmon sperm DNA. Filters are hybridized for 12 to 48 hrs at65° C. in prehybridization mixture containing 100 μg/ml denatured salmonsperm DNA and 5–20×10⁶ cpm of ³²P-labeled probe. Washing of filters isdone at 37° C. for 1 hr in a solution containing 2×SSC, 0.1% SDS. Thisis followed by a wash in 0.1×SSC, 0.1% SDS at 50° C. for 45 min. beforeautoradiography.

Other procedures using conditions of high stringency would includeeither a hybridization step carried out in 5×SSC, 5× Denhardt'ssolution, 50% formamide at 42° C. for 12 to 48 hours or a washing stepcarried out in 0.2×SSPE, 0.2% SDS at 65° C. for 30 to 60 minutes.

Reagents mentioned in the foregoing procedures for carrying out highstringency hybridization are well known in the art. Details of thecomposition of these reagents can be found in, e.g., Sambrook, et al.,1989, Molecular Cloning: A Laboratory Manual, second edition, ColdSpring Harbor Laboratory Press. In addition to the foregoing, otherconditions of high stringency which may be used are well known in theart.

Polypeptides

A preferred aspect of the present invention is a substantially purifiedform of the MurC protein from Pseudomonas aeruginosa. A preferredembodiment is a protein that has the amino acid sequence which is shownin FIG. 1, in SEQ ID NO:2 and disclosed as follows:

MetProAlaTrpArgTrpProValValLysGluProAsnGlyValThrArgThrMetArg (SEQ IDNO:2) ArgIleArgArgIleHisPheValGlyIleGlyGlyAlaGlyMetCysGlyIleAlaGluValLeuLeuAsnLeuGlyTyrGluValSerGlySerAspLeuLysAlaSerAlaValThrGluArgLeuGluLysPheGlyAlaGlnIlePheIleGlyHisGlnAlaGluAsnAlaAspGlyAlaAspValLeuValValSerSerAlaIleAsnArgAlaAsnProGluValAlaSerAlaLeuGluArgArgIleProValValProArgAlaGluMetLeuAlaGluLeuMetArgTyrArgHisGlyIleAlaValAlaGlyThrHisGlyLysThrThrThrThrSerLeuIleAlaSerValPheAlaAlaGlyGlyLeuAspProThrPheValIleGlyGlyArgLeuAsnAlaAlaGlyThrAsnAlaGlnLeuGlyAlaSerArgTyrLeuValAlaGluAlaAspGluSerAspAlaSerPheLeuHisLeuGlnProMetValAlaValValThrAsnIleAspAlaAspHisMetAlaThrTyrGlyGlyAspPheAsnLysLeuLysLysThrPheValGluPheLeuHisAsnLeuProPheTyrGlyLeuAlaValMetCysValAspAspProValValArgGluIleLeuProGlnIleAlaArgProThrValThrTyrGlyLeuSerGluAspAlaAspValArgAlaIleAsnIleArgGlnGluGlyMetArgThrTrpPheThrValLeuArgProGluArgGluProLeuAspValSerValAsnMetProGlyLeuHisAsnValLeuAsnSerLeuAlaThrIleValIleAlaThrAspGluGlyIleSerAspGluAlaIleValGlnGlyLeuSerGlyPheGlnGlyValGlyArgArgPheGlnValTyrGlyGluLeuGlnValGluGlyGlySerValMetLeuValAspAspTyrGlyHisHisProArgGluValAlaAlaValIleLysAlaIleArgGlyGlyTrpProGluArgArgLeuValMetValTyrGlnProHisArgTyrThrArgThrArgAspLeuTyrGluAspPheValGlnValLeuGlyGluAlaAsnValLeuLeuLeuMetGluValTyrProAlaGlyGluGluProIleProGlyAlaAspSerArgGlnLeuCysHisSerIleArgGlnArgGlyGlnLeuAspProIleTyrPheGluArgAspAlaAspLeuAlaProLeuValLysProLeuLeuArgAlaGlyAspIleLeuLeuCysGlnGlyAlaGlyAspValGlyGlyLeuAlaProGlnLeuIleLysAsnProLeuPheAlaGlyLysGlyGlyLysGlyAla

The present invention also relates to biologically active fragments andmutant or polymorphic forms of MurC polypeptide sequence as set forth asSEQ ID NO: 2, including but not limited to amino acid substitutions,deletions, additions, amino terminal truncations and carboxy-terminaltruncations such that these mutations provide for proteins or proteinfragments of diagnostic, therapeutic or prophylactic use and would beuseful for screening for modulators, and/or inhibitors of MurC function.

Using the disclosure of polynucleotide and polypeptide sequencesprovided herein to isolate polynucleotides encoding naturally occurringforms of MurC, one of skill in the art can determine whether suchnaturally occurring forms are mutant or polymorphic forms of MurC bysequence comparison. One can further determine whether the encodedprotein, or fragments of any MurC protein, is biologically active byroutine testing of the protein of fragment in a in vitro or in vivoassay for the biological activity of the MurC protein. For example, onecan express N-terminal or C-terminal truncations, or internal additionsor deletions, in host cells and test for their ability to catalyze theATP-dependent addition of L-alanine to the UDP-N-acetylmuramylprecursor.

It is known that there is a substantial amount of redundancy in thevarious codons which code for specific amino acids. Therefore, thisinvention is also directed to those DNA sequences that encode RNAcomprising alternative codons which code for the eventual translation ofthe identical amino acid.

Therefore, the present invention discloses codon redundancy which canresult in different DNA molecules encoding an identical protein. Forpurposes of this specification, a sequence bearing one or more replacedcodons will be defined as a degenerate variation. Also included withinthe scope of this invention are mutations either in the DNA sequence orthe translated protein which do not substantially alter the ultimatephysical properties of the expressed protein. For example, substitutionof valine for leucine, arginine for lysine, or asparagine for glutaminemay not cause a change in functionality of the polypeptide. However, anygiven change can be examined for any effect on biological function bysimply assaying for the ability to catalyze the ATP-dependent additionof L-alanine to an alanyl residue of the UDP-N-acetylmuramyl precursoras compared to an unaltered MurC protein.

It is known that DNA sequences coding for a peptide can be altered so asto code for a peptide having properties that are different than those ofthe naturally occurring peptide. Methods of altering the DNA sequencesinclude but are not limited to site directed mutagenesis. Examples ofaltered properties include but are not limited to changes in theaffinity of an enzyme for a substrate.

As used herein, a “biologically active equivalent” or “functionalderivative” of a wild-type MurC possesses a biological activity that issubstantially similar to the biological activity of a wild type MurC.The term “functional derivative” is intended to include the “fragments,”“mutants,” “variants,” “degenerate variants,” “analogs,” “orthologues,”and “homologues” and “chemical derivatives” of a wild type MurC proteinthat can catalyze the ATP-dependent addition of L-alanine to theUDP-N-acetylmuramyl precursor.

The term “fragment” refers to any polypeptide subset of wild-type MurC.The term “mutant” is meant to refer to a molecule that may besubstantially similar to the wild-type form but possesses distinguishingbiological characteristics. Such altered characteristics include but arein no way limited to altered substrate binding, altered substrateaffinity and altered sensitivity to chemical compounds affectingbiological activity of the MurC or MurC functional derivative. The term“variant” refers to a molecule substantially similar in structure andfunction to either the entire wild-type protein or to a fragmentthereof. A molecule is “substantially similar” to a wild-type MurC-likeprotein if both molecules have substantially similar structures or ifboth molecules possess similar biological activity. Therefore, if thetwo molecules possess substantially similar activity, they areconsidered to be variants even if the exact structure of one of themolecules is not found in the other or even if the two amino acidsequences are not identical. The term “analog” refers to a moleculesubstantially similar in function to either the full-length MurC proteinor to a biologically active fragment thereof.

As used herein in reference to a MurC gene or encoded protein, a“polymorphic” MurC is a MurC that is naturally found in the populationof Pseudomonads at large. A polymorphic form of MurC can be encoded by adifferent nucleotide sequence from the particular murC gene disclosedherein as SEQ ID NO:1. However, because of silent mutations, apolymorphic murC gene can encode the same or different amino acidsequence as that disclosed herein. Further, some polymorphic forms MurCwill exhibit biological characteristics that distinguish the form fromwild-type MurC activity, in which case the polymorphic form is also amutant.

A protein or fragment thereof is considered purified or isolated when itis obtained at least partially free from it's natural environment in acomposition or purity not found in nature. It is preferred that themolecule be present at a concentration at least about five-fold toten-fold higher than that found in nature. A protein or fragment thereofis considered substantially pure if it is obtained at a concentration ofat least about 100-fold higher than that found in nature. A protein orfragment thereof is considered essentially pure if it is obtained at aconcentration of at least about 1000-fold higher than that found innature. We most prefer proteins that have been purified to homogeneity,that is, at least 10,000–100,000 fold.

Probes and Primers

Polynucleotide probes comprising full length or partial sequences of SEQID NO: 1 can be used to determine whether a cell or sample contains P.aeruginosa MurC DNA or RNA. The effect of modulators that effect thetranscription of the murC gene can be studied via the use of theseprobes. A preferred probe is a single stranded antisense probe having atleast the full length of the coding sequence of MurC. It is alsopreferred to use probes that have less than the full length sequence,and contain sequences specific for P. aeruginosa murC DNA or RNA. Theidentification of a sequence(s) for use as a specific probe is wellknown in the art and involves choosing a sequence(s) that is unique tothe target sequence, or is specific thereto. It is preferred thatpolynucleotides that are probes have at least about 25 nucleotides, morepreferably about 30 to 35 nucleotides. The longer probes are believed tobe more specific for P. aeruginosa murC gene(s) and RNAs and can be usedunder more stringent hybridization conditions. Longer probes can be usedbut can be more difficult to prepare synthetically, or can result inlower yields from a synthesis. Examples of sequences that are useful asprobes or primers for P. aeruginosa murC gene(s) are Primer A (sense)

5′ TTCATATGCCTGCCTGGAGGTG 3′ (SEQ ID NO:3)and Primer B (antisense)

5′ TTGGATCCTCATGCGCCCTTCCCTCCCTTG 3′ (SEQ ID NO:4).These primers are nucleotides 55–76 (A) and the complement ofnucleotides 1442–1464 (B) respectively, of SEQ ID NO:1. Restrictionsites, underlined, for NdeI and BamHI are added to the 5′ ends of theprimers to allow cloning between the NdeI and BamHI sites of theexpression vector pET-15b. However, one skilled in the art willrecognize that these are only a few of the useful probe or primersequences that can be derived from SEQ ID NO:1.

Polynucleotides having sequences that are unique or specific for P.aeruginosa murC can be used as primers in amplification reaction assays.These assays can be used in tissue typing as described herein.Additionally, amplification reactions employing primers derived from P.aeruginosa murC sequences can be used to obtain amplified P. aeruginosamurC DNA using the murC DNA of the cells as an initial template. ThemurC DNA so obtained can be a mutant or polymorphic form of P.aeruginosa murC that differs from SEQ ID NO:1 by one or more nucleotidesof the MurC open reading frame or sequences flanking the ORF. Thedifferences can be associated with a non-defective naturally occurringform or with a defective form of MurC. Thus, polynucleotides of thisinvention can be used in identification of various polymorphic P.aeruginosa murC genes or the detection of an organism having a P.aeruginosa murC gene. Many types of amplification reactions are known inthe art and include, without limitation, Polymerase Chain Reaction,Reverse Transcriptase Polymerase Chain Reaction, Strand DisplacementAmplification and Self-Sustained Sequence Reaction. Any of these or likereactions can be used with primers derived from SEQ ID NO:1.

Expression of MurC

A variety of expression vectors can be used to express recombinant MurCin host cells. Expression vectors are defined herein as nucleic acidsequences that include regulatory sequences for the transcription ofcloned DNA and the translation of their mRNAs in an appropriate host.Such vectors can be used to express a bacterial gene in a variety ofhosts such as bacteria, bluegreen algae, plant cells, insect cells andanimal cells. Specifically designed vectors allow the shuttling of genesbetween hosts such as bacteria-yeast or bacteria-animal cells. Anappropriately constructed expression vector should contain: an origin ofreplication for autonomous replication in host cells, selectablemarkers, a limited number of useful restriction enzyme sites, apotential for high copy number, and regulatory sequences. A promoter isdefined as a regulatory sequence that directs RNA polymerase to bind toDNA and initiate RNA synthesis. A strong promoter is one which causesmRNAs to be initiated at high frequency. Expression vectors can include,but are not limited to, cloning vectors, modified cloning vectors,specifically designed plasmids or viruses.

In particular, a variety of bacterial expression vectors can be used toexpress recombinant MurC in bacterial cells. Commercially availablebacterial expression vectors which are suitable for recombinant MurCexpression include, but are not limited to pQE (Qiagen), pET11a orpET15b (Novagen), lambda gt11 (Invitrogen), and pKK223-3 (Pharmacia).

Alternatively, one can express murC DNA in cell-freetranscription-translation systems, or murC RNA in cell-free translationsystems. Cell-free synthesis of MurC can be in batch or continuousformats known in the art.

One can also synthesize MurC chemically, although this method is notpreferred.

A variety of host cells can be employed with expression vectors tosynthesize MurC protein. These can include E. coli, Bacillus, andSalmonella. Insect and yeast cells can also be appropriate.

Following expression of MurC in a host cell, MurC polypeptides can berecovered. Several protein purification procedures are available andsuitable for use. MurC protein and polypeptides can be purified fromcell lysates and extracts, or from culture medium, by variouscombinations of, or individual application of methods includingultrafiltration, acid extraction, alcohol precipitation, saltfractionation, ionic exchange chromatography, phosphocellulosechromatography, lecithin chromatography, affinity (e.g., antibody orHis-Ni) chromatography, size exclusion chromatography, hydroxylapatiteadsorption chromatography and chromatography based on hydrophobic orhydrophillic interactions. In some instances, protein denaturation andrefolding steps can be employed. High performance liquid chromatography(HPLC) and reversed phase HPLC can also be useful. Dialysis can be usedto adjust the final buffer composition.

The MurC protein itself is useful in assays to identify compounds thatmodulate the activity of the protein—including compounds that inhibitthe activity of the protein. The MurC protein is also useful for thegeneration of antibodies against the protein, structural studies of theprotein, and structure/function relationships of the protein.

Modulators and Inhibitors of MurC

The present invention is also directed to methods for screening forcompounds which modulate or inhibit a MurC protein. Compounds whichmodulate or inhibit MurC can be DNA, RNA, peptides, proteins, ornon-proteinaceous organic or inorganic compounds or other types ofmolecules. Compounds that modulate the expression of DNA or RNA encodingMurC or are inhibitors of the biological function of MurC can bedetected by a variety of assays. The assay can be a simple “yes/no”assay to determine whether there is a change in expression or function.The assay can be made quantitative by comparing the expression orfunction of a test sample with the levels of expression or function in astandard sample, that is, a control. A compound that is a modulator canbe detected by measuring the amount of the MurC produced in the presenceof the compound. An compound that is an inhibitor can be detected bymeasuring the specific activity of the MurC protein in the presence andabsence of the compound.

The proteins, DNA molecules, RNA molecules and antibodies lendthemselves to the formulation of kits suitable for the detection andanaysis of MurC. Such a kit would comprise a compartmentalized carriersuitable to hold in close confinement at least one container. Thecarrier would further comprise reagents such as recombinant MurC oranti-MurC antibodies suitable for detecting MurC. The carrier can alsocontain a means for detection such as labeled antigen or enzymesubstrates or the like.

Pharmaceutical Compositions

Pharmaceutically useful compositions comprising a modulator or inhibitorof MurC can be formulated according to known methods such as by theadmixture of a pharmaceutically acceptable carrier. Examples of suchcarriers and methods of formulation can be found in Remington'sPharmaceutical Sciences. To form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of the inhibitor.

Therapeutic, prophylactic or diagnostic compositions of the inventionare administered to an individual in amounts sufficient to treat,prevent or diagnose disorders. The effective amount can vary accordingto a variety of factors such as the individual's condition, weight, sexand age. Other factors include the mode of administration. Theappropriate amount can be determined by a skilled physician.

The pharmaceutical compositions can be provided to the individual by avariety of routes such as subcutaneous, topical, oral and intramuscular.

The term “chemical derivative” describes a molecule that containsadditional chemical moieties which are not normally a part of the basemolecule. Such moieties can improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties canattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Examples of such moieties are describedin a variety of texts, such as Remington's Pharmaceutical Sciences.

Compounds identified according to the methods disclosed herein can beused alone at appropriate dosages. Alternatively, co-administration orsequential administration of other agents can be desirable.

The present invention also provides a means to obtain suitable topical,oral, systemic and parenteral pharmaceutical formulations for use in themethods of treatment of the present invention. The compositionscontaining compounds identified according to this invention as theactive ingredient can be administered in a wide variety of therapeuticdosage forms in conventional vehicles for administration. For example,the compounds can be administered in such oral dosage forms as tablets,capsules (each including timed release and sustained releaseformulations), pills, powders, granules, elixirs, tinctures, solutions,suspensions, syrups and emulsions, or by injection. Likewise, they canalso be administered in intravenous (both bolus and infusion),intraperitoneal, subcutaneous, topical with or without occlusion, orintramuscular form, all using forms well known to those of ordinaryskill in the pharmaceutical arts.

Advantageously, compounds of the present invention can be administeredin a single daily dose, or the total daily dosage can be administered individed doses of two, three or four times daily. Furthermore, compoundsfor the present invention can be administered in intranasal form viatopical use of suitable intranasal vehicles, or via transdermal routes,using those forms of transdermal skin patches well known to those ofordinary skill in that art. To be administered in the form of atransdermal delivery system, the dosage administration will, of course,be continuous rather than intermittent throughout the dosage regimen.

For combination treatment with more than one active agent, where theactive agents are in separate dosage formulations, the active agents canbe administered concurrently, or they each can be administered atseparately staggered times.

The dosage regimen utilizing the compounds of the present invention isselected in accordance with a variety of factors including type,species, age, weight, sex and medical condition of the patient; theseverity of the condition to be treated; the route of administration;the renal, hepatic and cardiovascular function of the patient; and theparticular compound thereof employed. A physician or veterinarian ofordinary skill can readily determine and prescribe the effective amountof the drug required to prevent, counter or arrest the progress of thecondition. Optimal precision in achieving concentrations of drug withinthe range that yields efficacy without toxicity requires a regimen basedon the kinetics of the drug's availability to target sites. Thisinvolves a consideration of the distribution, equilibrium, andelimination of a drug.

The following examples are presented by the way of illustration and,because various other embodiments will be apparent to those in the art,the following is not to be construed as a limitation on the scope of theinvention. For example, while particular preferred embodiments of theinvention are presented herein, it is within the ability of persons ofordinary skill in the art to modify or substitute vectors, host cells,compositions, etc., or to modify or design protocols or assays, all ofwhich may reach the same or equivalent performance or results as theembodiments shown herein.

EXAMPLE 1

General Materials and Methods

All reagents were purchased from SIGMA CHEMICAL CO., St. Louis, Mo.,unless otherwise indicated. UDP-N-acetylmuramyl-L-alanine wassynthesized and purified by a method known in the art (Jin, H.,Emanuele, J. J., Jr., Fairman, R., Robertson, J. G., Hail, M. E., Ho,H.-T., Falk, P. and Villafranca, J. J, 1996. Structural studies ofEscherichia coli UDP-N-acetylmuramate: L-alanine ligase, Biochemistry35: 14423–14431).

DNA Manipulations Reagents and Techniques.

Restriction endonucleases and T4 ligase were obtained from GIBCO-BRL.Agarose gel electrophoresis and plasmid DNA preparations were performedaccording to published procedures (Sambrook, J., E. F. Fritsch, and T.Maniatis, 1989, Molecular cloning: a L, Laboratory Manual, 2nd ed. ColdSpring Harbor, NY: Cold Spring Harbor Laboratory). Recombinant plasmidscontaining P. aeruginosa murC were propagated in E. coli DH5a(GIBCO-BRL, Rockville, Md.) prior to protein expression in E. coliBL21(DE3)/plysS (NOVAGEN, Madison, Wis.). SDS-PAGE was performed withprecast gels (NOVAGEN). DNA sequences were determined using an automatedABI PRISM™ DNA sequencer (PERKIN-ELMER ABI, Foster City, Calif.).

EXAMPLE 2

Cloning of Pseudomonas aeruginosa murC

Genomic DNA from P. aeruginosa (strain MB4439) was prepared from 100 mllate stationary phase culture in Brain Heart Infusion broth (DIFCO,Detroit, Mich.). Cells were washed with 0.2 M sodium acetate, suspendedin 10 ml of TEG (100 mM Tris, pH 7, containing 10 mM EDTA and 25%glucose) and lysed by incubation with 200 μg of N-acetylmuramidase(SIGMA) for 1 h at 37° C. Chromosomal DNA was purified from the celllysate using a QIAGEN (Santa Clarita, Calif.) genomic DNA preparationkit and following the manufacturers protocol. Briefly, the cell lysatewas treated with protease K at 50° C. for 45 min, loaded onto anequilibrated QIAGEN genomic tip, entered into the resin bycentrifugation at 3000 rpm for 2 min. Following washing the genomic tip,the genomic DNA was eluted in distilled water and kept at 4° C.Approximately 50 ng genomic DNA was used as a template in PCR reactionsto clone murC.

Two oligonucleotide primers (GIBCO/BRL, Bethesda, Md.) complementary tosequences at the 5′ and the 3′ ends of P. aeruginosa murC were used toclone this gene using KLENTAQ ADVANTAGE™ polymerase (CLONTECH, PaloAlto, Calif.). The primer nucleotide sequences were as follows:

5′-TTCATATGCCTGCCTGGAGGTG-3′ (SEQ ID NO:3)(a NdeI linker within nucleotides 55–76 of SEQ ID NO: 1) and

5′-TTGGATCCTCATGCGCCCTTCCCTCCCTTG-3′ (SEQ ID NO:4)(a BamHI linker within the complement of nucleotides 1442–1464 of SEQ IDNO: 1). A PCR product representing P. aeruginosa murC was verified bynucleotide sequence, digested with NdeI and BamHI, and cloned betweenthe NdeI and BamHI sites of pET-15b, creating plasmid pPaeMurC. Thisplasmid was used for expression of the murC gene in E. coli.

EXAMPLE 3

Sequence Analysis of Pseudomonas aeruginosa murC

The nucleotide sequence of murC, determined in both orientations, andthe deduced amino acid sequence of the MurC protein is depicted in FIGS.1A–1B. Sequence comparison using the BLAST algorithm (Altschul, S. F.,Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) “Basic localalignment search tool.” J. Mol. Biol. 215:403–410) against the GenBankdatabase showed that, to varying degrees, the cloned region ishomologous (76% similar, 59% identical) to murC gene from E. coli(Ikeda, M., Wachi, M., Jung, H. K., Ishino, F. and Matsuhashi, M. 1990.Nucleotide sequence involving murG and murC in the mra gene cluster ofEscherichia coli. Nucleic Acids Res. 18:4014).

EXAMPLE 4

Overexpression, Purification and Enzymatic Activity of Pseudomonasaeruginosa MurC

murC was cloned into the expression vector pET-15b (NOVAGEN) asdescribed above to create plasmid pPaeMurC. The pET-15b vectorincorporates the 6× Histidine-tag into the protein construct to allowrapid purification of MurC by affinity chromatography. The pET (Plasmidsfor Expression by T7 RNA polymerase) plasmids are derived from pBR322and designed for protein over-production in E. coli. The vector pET-15bcontains the ampicillin resistance gene, ColE1 origin of replication inaddition to T7 phage promoter and terminator. The T7 promoter isrecognized by the phage T7 RNA polymerase but not by the E. coli RNApolymerase. A host E coli strain such as BL21(DE3)pLysS is engineered tocontain integrated copies of T7 RNA polymerase under the control oflacUV5 that is inducible by IPTG. Production of a recombinant protein inthe E. coli strain BL21(DE3)pLysS occurs after expression of T7RNApolymerase is induced.

The pPaeMurC plasmid was introduced into the host strain BL21 DE3/pLysS(NOVAGEN) for expression of His-tagged MurC. Colonies were grown at 37°C. in 100 ml of LB broth containing 100 mg/ml ampicillin and 32 μg/mlchloramphenicol. When cultures reached a cell density of A₆₀₀=0.5, cellswere pelleted and then resuspended in M9ZB medium (NOVAGEN) containing 1mM IPTG. Cells were induced for 3 h at 30° C., pelleted at 3000 g, andfrozen at −80° C.

Cultures containing either the recombinant plasmid pPaeMurC or thecontrol plasmid vector, pET-15b were grown at 30° C. and induced withIPTG. Cells transformed with pPaeMurC contained an inducible protein ofapproximately 54.7 kDa, corresponding to the expected size of P.aeruginosa MurC protein as shown by SDS-PAGE. (FIG. 2.) There were nocomparable detectable protein bands after induction of cells transformedwith the control plasmid vector, pET-15b.

Purification of Recombinant MurC Enzyme

The cell pellet from 100 ml of induced culture prepared as describedabove was resuspended in 10 ml BT buffer (50 mM bis-tris-propane, pH8.0, containing 100 mM potassium chloride and 1% glycerol) at 4° C.Cells were lysed either by freeze-thaw or by French Press. Aftercentrifugation, the supernatant was mixed with 15 ml of freshly preparedTALON (CLONTECH) resin and incubated for 30 min at room temp. The resinwas washed twice by centrifugation with 25 ml of BT buffer at roomtemperature. Finally, the resin was loaded into a column and washed with20 ml of BT, pH 7.0, containing 5 mM imidazole. Protein was eluted with20 ml of BT buffer pH 8.0, containing 100 mM imidazole. Fractions (0.5ml) were collected and analyzed by SDS-Gel electrophoresis. (FIG. 2)This resulted in a partially purified preparation of P. aeruginosa MurCprotein that could be used in activity assays. The protein may bepurified further, if desired, using methods known in the art.

Assay for Activity of MurC Enzyme.

The ATP-dependent MurC activity was assayed by monitoring the formationof product ADP using the pyruvate kinase and lactate dehydrogenasecoupled enzyme assay. The reaction was monitored spectrophotometrically.

Typically, the assay contained 100 mM BIS-TRIS-propane, pH 8.0, 200 μMNADH, 1 mM ATP, 20 mM PEP, 5 mM MgCl₂, 1 mM DTT, 350 μMUDP-N-acetyl-muramyl, 1 mM L-alanine, 33 units/ml of pyruvate kinase and1660 units/ml of lactate dehydrogenase in a final volume of 200 or 400μl. The mixture was incubated at 25° C. for 5 min and the reactioninitiated by the addition of 1–10 μg of MurC. These conditions are oneexample of an assay useful for evaluating the activity of MurC. Otherassays can be used, or amounts of buffers, substrate and enzyme can bechanged, as desired, to alter the rate of production of ADP.

ADP formation was monitored by the decrease in absorbance at 340 nm as afunction of time using a MOLECULAR DEVICES SPECTRAMAXPLUS™microtiterplate spectrophotomer (for 200 μl assays) or a HEWLETT-PACKARDHP8452A spectrophotometer equipped with a circulating water bath (for400 μl assays). Rates were calculated from the linear portions of theprogress curves using the extinction coefficient for NADH, e=6220 cm⁻¹M⁻¹. One unit of MurC activity is equal to 1 μmol of ADP formed per minat 25° C. MurC activity co-eluted with a ∥51 kDa protein.

TABLE 1 Specific activities of recombinant MurC from E. coli and P.aeruginosa. P. aeruginosa E. coli Mur Ligase μmol × min⁻¹ × mg⁻¹ μmol ×min⁻¹ × mg⁻¹ MurC 0.3 0.066

EXAMPLE 5

Screening for Inhibitors of MurC

One assay for the measurement of the activity of MurC is provided inExample 4. That assay, and other assays for MurC activity can be adaptedfor screening assays to detect inhibitors of MurC. For example, forinhibition assays, inhibitors in DMSO are added at the desiredconcentration to the assay mixture. In a separate, control reaction,only DMSO is added to the assay mixture. The reactions are initiated bythe addition of enzyme (MurC). Rates are calculated as described above.Relative activities are calculated from the equation 1:relative activity=rate with inhibitor/rate without inhibitor.  (1)Inhibition constant (IC₅₀) values are determined from a range ofinhibitor concentrations and calculated from equation 2.relative activity=1/(1+[I]/IC ₅₀)  (2)

One can use computer software to assist in the analysis, e.g., SIGMAPLOT™ (JANDEL SCIENTIFIC, San Rafeal, Calif.).

We prefer inhibitors of MurC that result in relative activities of theMurC enzyme of at least less than 75%, more preferably, 25–50% or10–25%. We most prefer inhibitors resulting in relative activities ofless than 20%, particularly less than 10% of the activity of MurC in theabsence of the inhibitor.

We also prefer inhibitors that effectively lower the relative activityof MurC when the inhibitor is present at a very low concentration.

EXAMPLE 8

Therapy Using Inhibitors of MurC

A patient presenting with an indication of infection with amicroorganism susceptible to inhibitors of MurC, e.g., gram positive andnegative bacteria, including P. aeruginosa, can be treated byadministration of inhibitors of MurC. Physicians skilled in the art arefamiliar with administering therapeutically effective amounts ofinhibitors or modulators of microbial enzymes. Such skilled persons canreadily determine an appropriate dosing scheme to achieve a desiredtherapeutic effect.

Therapy can also be prophylactic. For example, a patient at risk fordeveloping a bacterial infection, including infection with P.aeruginosa, can be treated by administration of inhibitors of MurC.Physicians skilled in the art are familiar with administeringtherapeutically effective amounts of inhibitors or modulators ofmicrobial enzymes. Such skilled persons can readily determine anappropriate dosing scheme to achieve a desired therapeutic effect.

1. A purified and isolated polynucleotide encoding a polypeptide havingthe amino acid sequence of SEQ ID NO:
 2. 2. The polynucleotide of claim1 wherein the polynucleotide encoding the polypeptide of SEQ ID NO:2 isa DNA polynucleotide comprising the polynucleotide sequence of SEQ IDNO:1.
 3. A purified and isolated polynucleotide that is an expressionvector comprising the polynucleotide of claim
 1. 4. A purified andisolated host cell comprising the expression vector of claim
 3. 5. Aprocess for expressing a MurC polypeptide of Pseudomonas aeruginosa in arecombinant host cell, comprising: (a) transforming a suitable host cellwith the expression vector of claim 3, and, (b) culturing the host cellof step (a) in and under conditions which allow expression of said theMurC polypeptide from said expression vector.
 6. A purified and isolatedpolypeptide having the amino acid sequence of SEQ ID NO:
 2. 7. A methodof determining whether a candidate compound is an inhibitor of aPseudomonas aeruginosa MurC polypeptide comprising: (a) providing atleast one host cell harboring an expression vector that includes apolynucleotide encoding a polypeptide having the amino acid sequence ofSEQ ID NO: 2, and (b) culturing said host cell under conditions thatpromote the expression of said polypeptide, and (c) contacting said atleast one cell with the candidate to permit the interaction of thecandidate with the MurC polypeptide, and (d) determining whether thecandidate is an inhibitor of the MurC polypeptide by ascertaining theactivity of the polypeptide in the presence of the candidate.
 8. Themethod of claim 7 wherein the polynucleotide encoding a polypeptidehaving the amino acid sequence of SEQ ID NO: 2 has the polynucleotidesequence of SEQ ID NO:
 1. 9. The method of claim 7 wherein thedetermination of activity in step (d) comprises comparing a measurementof MurC polypeptide activity of said at least one cell before step (c)to a measurement of MurC polypeptide activity of said at least one cellafter step (c).
 10. A method of determining whether a candidate compoundis an inhibitor of a Pseudomonas aeruginosa MurC polypeptide comprising:(a) providing a sample that includes a MurC polypeptide having the aminoacid sequence of SEQ ID NO: 2, and (b) contacting said sample with thecandidate to permit the interaction of the candidate with the MurCpolypeptide, and (c) determining whether the candidate is an inhibitorof the MurC polypeptide by ascertaining the activity of the MurCpolypeptide in the presence of the candidate.
 11. The method of claim 10wherein in step (c) the activity is determined by comparing ameasurement of MurC polypeptide activity of the sample before step (b)to a measurement of MurC polypeptide activity of the sample after step(b).